Baumschabl, M.; Lutz, L.; Jecmenica, M.; Ata, O.; Mattanovich, D.

Synthetic autotrophs are a promising platform for sustainable bioproduction using CO2 as substrate. The methylotrophic yeast Komagataella phaffii has been engineered to use CO2 as the sole carbon source by integration of the Calvin-Benson-Bassham (CBB) cycle, based on its native methanol assimilating xylulose monophosphate pathway (XuMP) cycle. Initial growth rates were low, but could be doubled by adaptive laboratory evolution (ALE). Beneficial mutations led to a decrease of CBB cycle reactions, indicating further limitations. During this study, temperature was identified as one of the key process parameters to improve autotrophic growth. For this reason, a new round of adaptive laboratory evolution was performed at the identified optimal cultivation temperature of 25{degrees}C, resulting in isolates growing up to 50 % faster compared to the control strain. Whole genome resequencing followed by reverse engineering helped to identify first key mutations of the evolved strains. In addition, targeted engineering was performed by increasing the copy number of the key gene of the CBB cycle RuBisCO, which is the bottleneck of carbon fixation. Combining this with the optimal cultivation temperature boosted maximum specific growth rates of the autotrophic K. phaffii strain. In comparison to ALE, the targeted engineering still is lagging behind a bit. Starting from the initial condition, growth was boosted more than 2.5-fold in this study to a maximum of 0.025 h-1.